Desaturases and methods of using them for synthesis of polyunsaturated fatty acids

ABSTRACT

The amino acid and nucleic acid sequences of a Δ 5 -desaturase enzyme and a Δ 8 -desaturase enzyme are disclosed. The nucleic acid sequences can be used to design recombinant DNA constructs and vectors. These vectors can then be used to transform various organisms, including for example, plants and yeast. The transformed organisms will then produce polyunsaturated fatty acids. The amino acid sequences are useful for generating enzyme-specific antibodies that are useful for identifying the desaturases.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.13/406,248, filed Feb. 27, 2012, now U.S. Pat. No. 8,791,327; which is acontinuation of U.S. patent application Ser. No. 12/141,865, filed Jun.18, 2008, now U.S. Pat. No. 8,124,838 on Feb. 28, 2012; which is adivisional of U.S. patent application Ser. No. 10/975,692, filed Oct.26, 2004, now U.S. Pat. No. 7,402,735, issued Jul. 22, 2008; which is adivisional of U.S. patent application Ser. No. 09/857,583, filed Aug.17, 2001, now U.S. Pat. No. 6,825,017, issued Nov. 30, 2004; which isthe U.S. National Stage of International Application No. PCT/US99/28655,filed Dec. 6, 1999; and which claims the benefit of U.S. ProvisionalApplication No. 60/111,301, filed Dec. 7, 1998. All of these areincorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

This work was supported by funds from the U.S. Department of Agricultureunder NRICGP contract numbers 95-37301-2287 and 97-35301-4426. TheUnited States government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “Sequence_Listing.txt”, created on Mar. 19, 2014, and having asize of ˜28 kilobytes, as permitted under 37 CFR 1.821(c). The materialin the aforementioned file is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to desaturase enzymes that can be used to producepolyunsaturated fatty acids with important dietary applications.

BACKGROUND

Fatty acids are fundamental components of living systems. They make upthe major component of cytoplasmic membranes, common to plants, animalsand protists alike.

Fatty acids of 20 carbons, with more than one unsaturated carbon-carbonbond along the hydrocarbon chain, are known to be of particularimportance. Arachidonate (20:4) (Heinz, Lipid Metabolism in Plants, pp.33-89, 1993; Yamazaki et al. Biochim. Biophys. Acta 1123:18-26, 1992;Ulsamer et al., J. Cell Biol. 43:105-114, 1969; and Albert et al. Lipids14:498-500, 1979) and eicosapentaenoate (20:5) (Heinz, Lipid Metabolismin Plants, pp. 33-89, 1993; Yamazaki et al., Biochim. Biophys. Acta1123:18-26, 1992; Ulsamer et al., J. Cell Biol. 43:105-114, 1969; Albertet al. Lipids 14:498-500, 1979; and Cook et al., J. Lipid Res.32:1265-1273, 1991), commonly referred to as EPA, are significantcomponents of mammalian cell membranes and are also precursors of signalmolecules including prostaglandins. Certain specialized mammaliantissues such as brain (Naughton, J. Biochem. 13:21-32, 1981), testes(Wilder and Coniglio, Proc. Soc. Exp. Biol. Med. 177:399-405, 1984), andretina (Aveldano de Caldironi et al., Prog. Lipid Res. 20:49-57, 1981)are especially rich in unsaturated fatty acids.

Arachidonate and eicosapentaenoate serve both as precursors forsynthesis of 22-carbon polyunsaturated fatty acids and, withdihomo-γ-linoleate (20:3) (Yamazaki et al., Biochim. Biophys. Acta1123:18-26, 1992; Ulsamer et al., J. Cell Biol. 43:105-114, 1969; andAlbert et al., Lipids 14:498-500, 1979), as precursors to the synthesisof eicosanoid metabolic regulators (Hwang, Fatty Acids in Foods andTheir Health Implications, 545-557, 1992). Key enzymes in the synthesisof 20-carbon fatty acids are desaturases, which introduce cis doublebonds by removing two hydrogen atoms at specific locations along thealiphatic hydrocarbon chains. Desaturase enzymes are specific to theposition, number, and stereochemistry of the double bonds alreadypresent in the target fatty acid (Heinz, Lipid Metabolism in Plants,33-89, 1993).

To synthesize 20-carbon polyunsaturated fatty acids, mammals mustacquire the essential fatty acids 18:2 (Brenner, The Role of Fats inHuman Nutrition, pp. 45-79, 1989) and 18:3 (Nelson, Fatty Acids in Foodsand Their Health Implications, pp. 437-471, 1992; Brenner, The Role ofFats in Human Nutrition, pp. 45-79, 1989; and Hulanicka et al. J. Biol.Chem. 239:2778-2787, 1964) from their diet (Nelson, Fatty Acids in Foodsand Their Health Implications, 437-471, 1992). These dietarypolyunsaturated fatty acids are metabolized in the endoplasmic reticulumby an alternating series of position-specific desaturases andmalonyl-CoA-dependent chain-elongation steps (FIG. 1A), which results inthe characteristic methylene-interrupted double bond pattern. In theliver, which is the primary organ of human lipid metabolism, the firststep in biosynthesis of 20-carbon fatty acids is desaturation of theessential fatty acids at the Δ⁶ position. The desaturation products areelongated to 20:3 and 20:4 (Cook et al., J. Lipid Res. 32:1265-1273,1991). In turn, these 20-carbon products are desaturated by aΔ⁵-desaturase to produce arachidonate and eicosapentaenoate. TheΔ⁶-desaturation step is rate-limiting in this metabolic pathway (Bernetand Sprecher, Biochim. Biophys. Acta 398:354-363, 1975; and Yamazaki etal., Biochim. Biophys. Acta 1123:18-26, 1992) and, not surprisingly, issubject to regulation by dietary and hormonal changes (Brenner, The Roleof Fats in Human Nutrition, pp. 45-79, 1989).

In contrast to the liver, an alternate pathway for biosynthesis of20-carbon polyunsaturated fatty acids has been demonstrated in a feworganisms and tissues (FIG. 1B). Instead of desaturation, the first stepin the alternate pathway is elongation of the essential 18-carbon fattyacids to 20-carbon chain lengths, producing 20:2 (Ulsamer et al., J.Cell Biol. 43:105-114, 1969; and Albert et al. Lipids 14:498-500, 1979)and 20:3. Subsequent desaturation occurs via a Δ⁸-desaturase (FIG. 1).The products of this elongation-desaturation, 20:3 and 20:4, are thesame as the more usual desaturation-elongation pathway. The Δ⁸ pathwayis present in the soil amoebae Acanthamoeba sp. (Ulsamer et al, J. CellBiol. 43:105-114, 1969), and in euglenoid species, where it is thedominant pathway for formation of 20-carbon polyunsaturated fatty acids(Hulanicka et al., Journal of Biological Chemistry 239:2778-2787, 1964).

This Δ⁸-desaturation pathway occurs in mammals, both in rat testis(Albert and Coniglio, Biochim. Biophys. Acta 489:390-396, 1977) and inhuman testis (Albert et al., Lipids 14:498-500, 1979). While Δ⁸ activityhas been observed in breast cancer cell lines (Grammatikos et al., Br.J. Cancer 70:219-227, 1994; and Bardon et al., Cancer Lett. 99:51-58,1996) and in glioma (Cook et al., J. Lipid Res. 32:1265-1273, 1991), noΔ⁸ activity is detectable in a corresponding non-cancerous breast cellline (Grammatikos et al., Br. J. Cancer 70:219-227, 1994) or in thebrain (Dhopeshwarkar and Subramanian, J. Neurochem. 36:1175-1179, 1976).The significance of Δ⁸-desaturation to normal or cancer cell metabolismis unclear, since analysis of desaturase activities in mammalian systemsis frequently complicated by the presence of competing Δ⁶ reactions andchain-shortening retroconversion of fatty acid substrates in tissue(Sprecher and Lee, Biochim. Biophys. Acta 388:113-125, 1975; Geiger etal., Biochim. Biophys. Acta 1170:137-142, 1993).

Polyunsaturated 20-carbon fatty acids are, for the reasons outlinedabove, important in the human diet, and there has been considerablerecent interest in incorporating such fatty acids into infant food, babyformula, dietary supplements, and nutraceutical formulations.

It would therefore be desirable to produce new transgenic plants andanimals with enhanced ability to produce polyunsaturated 20-carbon fattyacids.

SUMMARY OF THE DISCLOSURE

The invention provides novel Δ⁵- (FIG. 6A) and Δ⁸- (FIG. 7A) desaturaseenzymes that may be cloned and expressed in the cells of variousorganisms, including plants, to produce 20-carbon polyunsaturated fattyacids. Expression of such fatty acids enhances the nutritional qualitiesof such organisms. For instance, oil-seed plants may be engineered toincorporate the Δ⁵- and Δ⁸-desaturases of the invention. Such oil-seedplants would produce seed-oil rich in polyunsaturated 20:3, 20:4, 20:5,22:4, and 22:5 fatty acids. Such fatty acids could be incorporatedusefully into infant formula, foods of all kinds, dietary supplements,nutraceutical, and pharmaceutical formulations.

The invention also provides proteins differing from the proteins of FIG.6A and FIG. 7A by one or more conservative amino acid substitutions.Also provided are proteins that exhibit “substantial similarity”(defined in the “Definitions” section) with the proteins of FIG. 6A andFIG. 7A.

The invention provides isolated novel nucleic acids that encode theabove-mentioned proteins, recombinant nucleic acids that include suchnucleic acids and cells, and plants and organisms containing suchrecombinant nucleic acids.

The novel Δ⁵- and Δ⁸-desaturase enzymes can be used individually, or inconjunction with one another, for instance in a metabolic pathway, toproduce polyunsaturated fatty acids, such as 20:3, 20:4, 20:5, 22:4, and22:5 fatty acids.

The scope of the invention also includes portions of nucleic acidsencoding the novel Δ⁵- and Δ⁸-desaturase enzymes, portions of nucleicacids that encode polypeptides substantially similar to these novelenzymes, and portions of nucleic acids that encode polypeptides thatdiffer from the proteins of FIG. 6A and FIG. 7A by one or moreconservative amino acid substitutions. Such portions of nucleic acidsmay be used, for instance, as primers and probes for research anddiagnostic purposes. Research applications for such probes and primersinclude the identification and cloning of related Δ⁵- and Δ⁸-desaturasesin other organisms including humans.

The invention also includes methods that utilize the Δ⁵- and/or theΔ⁸-desaturase enzymes of the invention. An example of this embodiment isa yeast or plant cell that carries genes for one or both desaturases ofthe invention and that, by virtue of these desaturases, is able toproduce arachidonic acid and/or EPA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a common pathway for synthesis of 20-carbonpolyunsaturated fatty acids that begins with Δ⁶ desaturation of18-carbon fatty acids followed by 2-carbon elongation, and then furtherdesaturation and elongation.

FIG. 1B shows an alternate pathway that begins with an elongation of18-carbon fatty acid to 20-carbon fatty acids, followed by Δ⁸desaturation and a second desaturation at the Δ⁵ position.

FIG. 1C shows alternate pathways for the synthesis of polyunsaturatedfatty acids using Δ⁵-, Δ⁶-, and Δ⁸-desaturases to produce arachidonicacid and EPA.

FIG. 2 shows gas chromatographic (GC) analysis of fatty acid methylesters from E. gracilis grown (heterotrophically) in the dark withsucrose as carbon source. Fatty acids were identified by comparison ofretention times with known standards. Significant peaks are numberedwith their retention times and proportion of the total fatty acidindicated.

FIG. 3 shows amino acid sequence similarities between the Euglena Δ⁸-desaturase protein (EFD1) (SEQ ID NO: 4) and the desaturase enzymes ofC. elegans. The deduced amino acid sequence of the EFD1 gene showssimilarity with the C. elegans Δ ⁶ (FAT-3) (SEQ ID NO: 14) and Δ⁵(FAT-4) (SEQ ID NO: 2) desaturases (Napier et al., Biochem. J.330:611-614, 1998). The similarity is strongest in the regions ofconserved function. In the N-terminal region amino acids forming acytochrome b₅-like domain (Lederer, Biochimie 76:674-692, 1994) areindicated. The His-box motifs indicated by underlined characters arepresent in other identified membrane desaturases (Napier et al.,Biochem. J. 330:611-614, 1998; Michaelson et al., J. Biol. Chem.273:19055-19059, 1998; and Shanklin and Cahoon, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 48:611-641, 1998).

FIG. 4 shows the results of gas chromatography of fatty acid methylesters from recombinant yeast. Cultures of yeast containing eithercontrol pYES2 or pYES2-541, which expresses the Euglena Δ ⁸-desaturase(EFD1) gene, were supplemented with the indicated 20-carbon fatty acids.The control strain does not desaturate the exogenous fatty acids. Forthe experimental strain, an arrow indicates the desaturation peak.

FIG. 5 shows the results of mass spectrometry (MS) of desaturationproducts. DMOX derivatives of EFD1 desaturation products were analyzedby GC-mass spectrometry. The molecular ion of each fatty acid is 2a.m.u. (atomic mass units) less than the substrate provided, as expectedfor insertion of a double bond. Desaturation at the Δ⁸ position isestablished by characteristic m/z peaks of 182 and 194 for each product,indicated by the bracket.

FIG. 6A shows the primary amino acid sequence of the fatty acidΔ⁵-desaturase from Caenorhabditis elegans (SEQ ID NO: 2).

FIG. 6B shows a nucleotide sequence including the ORF (open readingframe) that encodes the fatty acid Δ⁵-desaturase from Caenorhabditiselegans (SEQ ID NO: 1).

FIG. 7A shows the primary amino acid sequence of the fatty acidΔ⁸-desaturase from the protist Euglena gracilis (SEQ ID NO: 4).

FIG. 7B shows a nucleotide sequence including the ORF that encodes theΔ⁸-desaturase from the protist Euglena gracilis (SEQ ID NO: 3).

FIG. 8 shows certain features of the structure of the C. elegans Δ ⁵-and Δ⁶-desaturase genes. The relative location of gene products T13F2.1and W08D2.4 on their respective cosmids is shown above. The exonstructure of T13F2.1 (fat-4) and W08D2.4 (fat-3) showing the sites ofsequences encoding the SL1 splice site, the heme-binding motif ofcytochrome b₅ (cyt b₅), and the three conserved histidine box motifs(HBX) is shown below.

FIG. 9 shows a comparison of the predicted amino acid sequences of theborage Δ⁶-desaturase (bord6) (SEQ ID NO: 15), C. elegans FAT-3 (fat3)(SEQ ID NO: 14), C. elegans FAT-4 (fat4) (SEQ ID NO: 2), and theMortierella alpina Δ ⁵-(mord5) (SEQ ID NO: 16) desaturase. Identical orconserved residues are shaded, and the conserved HPGG heme-bindingdomain and the conserved histidine boxes are underlined. Abbreviations:bord6=Borago officinalis Δ ⁶-desaturase (GenBank accession numberU79010); fat4=C. elegans FAT-4 desaturase; fat3=C. elegans Δ ⁶desaturase sequence of W08D2.4 (GenBank accession number Z70271), editedto remove amino acids 38-67, on the basis of the cDNA sequence;mord5=Mortierella alpina Δ ⁵ desaturase (GenBank accession numberAF054824).

FIGS. 10A-10C show identification of arachidonic acid in transgenicyeast by gas chromatography-mass spectroscopy (GC-MS). Fatty acid methylesters of total lipids of S. cerevisiae grown for 16 hours underinducing conditions (2% galactose) supplemented with 0.2 mMdi-homo-γ-linolenic acid were analyzed by GC-MS. (A) Yeast transformedwith (empty) vector pYES2. (B) Yeast transformed with pYES2 vectorcarrying fat-4. The common peaks were identified as 16:0 (11.19-11.12min.), 16:1 (11.38 min.), 18:0 (13.07-13.08 min.), 18:1 (13.29 min.),20:3 (11.64-11.65 min.). The novel peaks are arachidonic acid (14.49min.) and 18:2 (12.91 min.). (C) The mass spectrum of the peak elutingat 14.49 min. This spectrum is indistinguishable from that of authenticmethyl-arachidonate.

FIGS. 11A and 11B show the novel desaturation products from substrateslacking a Δ⁸ double bond. (A) Partial GC trace of fatty acid methylesters derived from yeast expressing the fat-4 Δ⁵-desaturasesupplemented with 20:2Δ^(11,14) (14.81 min.). The desaturation productof this substrate elutes at 14.62 min. and has been identified as20:3Δ^(5,11,14). (B) Partial GC trace of yeast expressing the fat-4Δ⁵-desaturase supplemented with 20:3Δ^(11,14,17) (14.87 min.). Thedesaturation product of this substrate elutes at 14.69 min. and has beenidentified as 20:4Δ^(5,11,14,17).

FIG. 12 is a table comparing the substrate specificities of C. elegans Δ⁵- and Δ⁶-desaturases.

FIG. 13 is a table comparing incorporation and desaturation of fattyacids by yeast strains transformed with a control construct pYES, andwith pYES-541, a clone containing EGD1, the E. gracilis Δ ⁸-desaturasegene. S. cerevisiae strains containing a control vector (pYES) orexpressing EFD1 (pYES-541) were cultured in the presence of theindicated fatty acids. The cultures were harvested, washed, and methylesters prepared from total cells and analyzed by GC. The weight % oftotal fatty acid methyl esters is indicated.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three-letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood to be included by any reference to the displayed strand.

SEQ ID NO: 1 is the nucleotide sequence corresponding to the openreading frame of the fatty acid Δ⁵-desaturase from Caenorhabditiselegans.

SEQ ID NO: 2 is the primary amino acid sequence of the fatty acidΔ⁵-desaturase from Caenorhabditis elegans.

SEQ ID NO: 3 is the nucleotide sequence corresponding to the openreading frame of the fatty acid Δ⁸-desaturase from the protist Euglenagracilis.

SEQ ID NO: 4 is the primary amino acid sequence of fatty acidΔ⁸-desaturase from the protist Euglena gracilis.

SEQ ID NOs: 5-8 are primers used to amplify and clone theΔ⁸-desaturase-encoding nucleic acid sequence.

SEQ ID NO: 9 is a polyadenylation signal.

SEQ ID NO: 10 is a primer used to amplify and clone theΔ⁵-desaturase-encoding nucleic acid sequence.

SEQ ID NO: 11 is a short RNA leader sequence.

SEQ ID NO: 12 is the amino acid sequence of a histidine box motif.

SEQ ID NO: 13 is the amino acid sequence of a histidine box motif.

SEQ ID NO: 14 is the primary amino acid sequence of the fatty acidΔ⁶-desaturase from C. elegans.

SEQ ID NO: 15 is the primary amino acid sequence of the fatty acidΔ⁶-desaturase from Borago officinalis.

SEQ ID NO: 16 is the primary amino acid sequence of the fatty acidΔ⁵-desaturase from Mortierella alpina.

SEQ ID NO: 17 is the amino acid sequence of a histidine box motif.

DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes VI, Oxford University Press: New York, 1997. The nomenclature forDNA bases as set forth at 37 C.F.R. §1.822 is used. The standard one-and three-letter nomenclature for amino acid residues is used.

DEFINITIONS

Portion: A portion of a nucleic acid molecule is a stretch of contiguousnucleic acids corresponding to the sequence of that molecule that may beabout 15, 20, 30, 40, 50, or 60 nucleic acids in length. Such nucleotideportions may be used as probes or primers. A portion of a protein is astretch of contiguous amino acids corresponding to the amino acidsequence of that protein that may be about 5, 10, 20, 30, 40, or 50residues in length. As used herein, such a portion may correspond to anysegment of a nucleic acid molecule, for instance such a portion maycorrespond to a segment consisting of nucleotides 1-500, 501-1000, or1001-1451 of the sequence shown in FIG. 6B, or nucleotides 1-400,401-800, 801-1251 of the sequence shown in FIG. 7B.

Desaturase: A desaturase is an enzyme that promotes the formation ofcarbon-carbon double bonds in a hydrocarbon molecule.

Desaturase activity may be demonstrated by assays in which a preparationcontaining an enzyme is incubated with a suitable form of substratefatty acid and analyzed for conversion of this substrate to thepredicted fatty acid product. Alternatively, a DNA sequence proposed toencode a desaturase protein may be incorporated into a suitable vectorconstruct and thereby expressed in cells of a type that do not normallyhave an ability to desaturate a particular fatty acid substrate.Activity of the desaturase enzyme encoded by the DNA sequence can thenbe demonstrated by supplying a suitable form of substrate fatty acid tocells transformed with a vector containing the desaturase-encoding DNAsequence and to suitable control cells (for example, transformed withthe empty vector alone). In such an experiment, detection of thepredicted fatty acid product in cells containing the desaturase-encodingDNA sequence and not in control cells establishes the desaturaseactivity. Examples of this type of assay have been described in, forexample, Lee et al., Science 280:915-918, 1998; Napier et al., Biochem.J. 330:611-614, 1998; and Michaelson et al., J. Biol. Chem.273:19055-19059, 1998, which are incorporated herein by reference.

The Δ⁵-desaturase activity may be assayed by these techniques using, forexample, 20:3Δ^(8,11,14) as substrate and detecting 20:4Δ^(5,8,11,14) asthe product (Michaelson et al., J. Biol. Chem. 273:19005-19059, 1998).Other potential substrates for use in Δ⁵ activity assays include (butare not limited to) 10:2Δ^(11,14) (yielding 20:5Δ^(5,11,14) as theproduct) and 20:3Δ^(11,14,12) (yielding 20:4Δ^(5,11,14,17) as theproduct).

The Δ⁸-desaturase may be assayed by similar techniques using, forexample, 20:3Δ^(11,14,17) as the substrate and detecting20:4Δ^(8,11,14,17) as the product.

ORF: Open reading frame. An ORF is a contiguous series of nucleotidetriplets coding for amino acids. These sequences are usuallytranslatable into a peptide.

Homologs: Two nucleotide or amino acid sequences that share a commonancestral sequence and diverged when a species carrying that ancestralsequence split into two species. Homologs frequently show a substantialdegree of sequence identity.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. The termencompasses all techniques by which a nucleic acid molecule might beintroduced into such a cell, including transfection with viral vectors,transformation with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the subject protein or other substance ismore pure than in its natural environment within a cell. Generally, aprotein preparation is purified such that the protein represents atleast 50% of the total protein content of the preparation.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame. If introns are present, the operably linked DNA sequencesmay not be contiguous.

Cell: A plant, animal, protist, bacterial, or fungal cell.

Sequence similarity: The similarity between two nucleic acids or twoamino acid sequences is expressed in terms of percentage sequenceidentity. The higher the percentage sequence identity between twosequences, the more similar the two sequences are.

In the case of protein alignments, similarity is measured not only interms of percentage identity, but also takes into account conservativeamino acid substitutions. Such conservative substitutions generallypreserve the hydrophobicity and acidity of the substituted residue, thuspreserving the structure (and therefore function) of the folded protein.The computer programs used to calculate protein similarity employstandardized algorithms that, when used with standardized settings,allow the meaningful comparison of similarities between different pairsof proteins.

Sequences are aligned, with allowances for gaps in alignment, andregions of identity are quantified using a computerized algorithm.Default parameters of the computer program are commonly used to set gapallowances and other variables.

Methods of alignment of sequences for comparison are well-known in theart. Various programs and alignment algorithms are described by Pearsonet al., Methods in Molecular Biology 24: 307-331, 1994, and in Altschulet al., Nature Genet. 6:119-129, 1994. Altschul et al. presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NBCI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.BLAST™ can be assessed by the web site with the host name “www” and thedomain name of “ncbi.nlm.nih.gov”. A description of how to determinesequence identity using this program is available at the web site. Asused herein, sequence identity is commonly determined with the BLAST™software set to default parameters. For instance, blastn (version 2.0)software may be used to determine sequence identity between two nucleicacid sequences using default parameters (expect=10, matrix=BLOSUM62,filter=DUST (Tatusov and Lipmann, in preparation as of Dec. 1, 1999; andHancock and Armstrong, Comput. Appl. Biosci. 10:67-70, 1994), gapexistence cost=11, per residue gap cost=1, and lambda ratio=0.85). Forcomparison of two polypeptides, blastp (version 2.0) software may beused with default parameters (expect 10, filter=SEG (Wootton andFederhen, Computers in Chemistry 17:149-163, 1993), matrix=BLOSUM62, gapexistence cost=11, per residue gap cost=1, lambda=0.85).

When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties).

An alternative alignment tool is the ALIGN Global Optimal Alignment tool(version 3.0) available from Biology Workbench online atbiology.ncsa.uiuc.edu. This tool may be used with settings set todefault parameters to align two known sequences. References for thistool include Meyers and Miller, CABIOS 4:11-17, 1989.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, i.e.,the structure and especially the function of the protein is conservedand not significantly changed by such substitutions. The table belowshows amino acids that may be substituted for an original amino acid ina protein and that are regarded as conservative substitutions.

TABLE 1 Original Conservative Residue Substitutions ala ser arg lys asngln; his asp glu cys ser gln asn glu asp gly pro his asn; gln ile leu;val leu ile; val lys arg; gln; glu met leu; ile phe met; leu; tyr serthr thr ser trp tyr tyr trp; phe val ile; leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histadyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

Probe: An isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes.

Primers: Short nucleic acids, preferably DNA oligonucleotides 10nucleotides or more in length, that are annealable to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extendable along the targetDNA strand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides. In order to enhance specificity,longer probes and primers may also be employed, such as probes andprimers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or150 consecutive nucleotides of the disclosed nucleic acid sequences.

Alternatively, such probes and primers may comprise at least 15, 20, 30,40, 50, 60, 70, 80, 90, 100, or 150 consecutive nucleotides that share adefined level of sequence identity with one of the disclosed sequences,for instance, at least a 50%, 60%, 70%, 80%, 90%, or 95% sequenceidentity.

Alternatively, such probes and primers may be nucleotide molecules thathybridize under specific conditions and remain hybridized under specificwash conditions such as those provided below. These conditions can beused to identifying variants of the desaturases. Nucleic acid moleculesthat are derived from the desaturase cDNA and gene sequences includemolecules that hybridize under various conditions to the discloseddesaturase nucleic acid molecules, or fragments thereof. Generally,hybridization conditions are classified into categories, for examplevery high stringency, high stringency, and low stringency. Theconditions for probes that are about 600 base pairs or more in lengthare provided below in three corresponding categories.

Very High Stringency (Detects Sequences that Share 90% SequenceIdentity)

Hybridization in SSC at 65° C. 16 hours Wash twice in SSC at room temp.15 minutes each Wash twice in SSC at 65° C. 20 minutes eachHigh Stringency (Detects Sequences that Share 80% Sequence Identity orGreater)

Hybridization in SSC at 65° C.-70° C. 16-20 hours Wash twice in SSC atroom temp. 5-20 minutes each Wash twice in SSC at 55° C.-70° C. 30minutes eachLow Stringency (Detects Sequences that Share Greater than 50% SequenceIdentity)

Hybridization in SSC at room temp.- 55° C. 16-20 hours Wash at least inSSC at room temp.- 55° C. 20-30 minutes twice each

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook et al. Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory Press, NY, 1989;Ausubel et al. Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley-Intersciences, 1987; and Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.,San Diego, Calif. 1990. PCR primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer (Version 0.5, 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Recombinant nucleic acid: A sequence that is not naturally occurring orhas a sequence that is made by an artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques such as those described in Sambrook etal. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press, NY, 1989. The term recombinant includes nucleicacids that have been altered solely by addition, substitution, ordeletion of a portion of the nucleic acid.

Native: The term “native” refers to a naturally-occurring (“wild-type”)nucleic acid or polypeptide. The native nucleic acid or protein may havebeen physically derived from a particular organism in which it isnaturally occurring or may be a synthetically constructed nucleic acidor protein that is identical to the naturally-occurring nucleic acid orprotein.

Isolated: An “isolated” nucleic acid is one that has been substantiallyseparated or purified away from other nucleic acid sequences in the cellof the organism in which the nucleic acid naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, by conventional nucleicacid-purification methods. The term also embraces recombinant nucleicacids and chemically synthesized nucleic acids.

Plant: The term “plant” encompasses any higher plant and progenythereof, including monocots (e.g., corn, rice, wheat, barley, rapeseed,soy, sunflower, etc.), dicots (e.g., potato, tomato, etc.), and includesparts of plants, including seeds, fruit, tubers, etc.

The invention will be better understood by reference to the Examplesherein. The scope of the invention is not to be considered limitedthereto.

Description and General Methods of the Disclosure

The present invention utilizes standard laboratory practices for thecloning, manipulation, and sequencing of nucleic acids, the purificationand analysis of proteins, and other molecular biological and biochemicaltechniques, unless otherwise stipulated. Such techniques are explainedin detail in standard laboratory manuals such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, NY, 1989; and Ausubel et al., Current Protocols inMolecular Biology, Green and Wiley-Interscience, NY, 1987.

The inventors have identified, cloned, and expressed a novel fatty acidΔ⁸-desaturase from the protist Euglena gracilis and a novel fatty acidΔ⁵-desaturase from Caenorhabditis elegans that may be used together toproduce polyunsaturated fatty acids.

The invention provides novel purified Δ⁵ and Δ⁸ proteins (FIG. 6A andFIG. 7A, respectively). The invention also provides proteins differingfrom the proteins of FIG. 6A and FIG. 7A by one or more conservativeamino acid substitutions, as well as proteins that show “substantialsimilarity” with the proteins of FIG. 6A and FIG. 7A. Substantialsimilarity is defined in the “Definitions” section. Proteins of theinvention include proteins that show at least 50% amino acid similaritywith the proteins shown in FIG. 6A and FIG. 7A. The term “50% amino acidsimilarity” is objectively and consistently defined by use of blastpsequence analysis software set at default parameters. Proteins of theinvention also include proteins showing at least 60%, at least 70%, atleast 80%, at least 90%, and at least 95% similarity (to the sequencesof FIG. 6A or FIG. 7A) using blastp with default parameters.

The invention provides isolated novel nucleic acids that encode theabove-mentioned proteins, recombinant nucleic acids that include suchnucleic acids and cells containing such recombinant nucleic acids.Nucleic acids of the invention thus include nucleic acids that encode:(1) amino acid sequences as shown in FIG. 6A and FIG. 7A; (2) amino acidsequences that differ from the sequences shown in FIG. 6A and FIG. 7A byone or more conservative amino acid substitutions; and (3) amino acidsequences that show at least 50% similarity (as measured by blastp atdefault parameters) with the sequence of FIG. 6A and FIG. 7A.

Nucleic acids of the invention also include nucleic acids that show atleast the term “50% similarity” with the nucleic acids shown in FIG. 6Band FIG. 7B. The term “50% similarity” is objectively defined by the useof blastn software set at default perimeters. Nucleic acids of theinvention also include nucleic acids showing at least 60%, at least 70%,at least 80%, at least 90%, and at least 95% similarity (to thesequences of FIG. 6B and FIG. 7B) using blastn with default perimeters.

The novel Δ⁵- and Δ⁸-desaturase enzymes can be used individually, or inconjunction with one another, for instance in a metabolic pathway, toproduce polyunsaturated fatty acids, such as 20:3 and 20:4 fatty acids.FIG. 1B shows an example of such a metabolic pathway. Such a pathway maybe engineered into any cell by use of appropriate expression systems. Asimple way to provide such elements is by the use of commerciallyavailable expression systems, discussed in detail below.

The scope of the invention covers not only entire nucleic acids encodingthe novel Δ⁵- and Δ⁸ desaturase enzymes (and substantially similarderivatives of such enzymes) but also covers “portions” of such nucleicacids (as defined in the “Definitions” section, herein). Such claimedportions are identified by their possession of a particular degree ofsimilarity with similar sized portions of the nucleotides of FIG. 6B andFIG. 7B and may have a length of about 15, 20, 30, 40, or 50 contiguousnucleotides. Similarity is objectively measured by sequence comparisonsoftware, such as the “blastn” and “blastp” software available from theNational Center for Biotechnology Information (NBCI, Bethesda, Md.) andon the Internet at the web site with the host name “www” and the domainname of “ncbi.nlm.nih.gov”. Similarity between portions of nucleic acidsclaimed and similar sized portions of the nucleic acid sequences of FIG.6B and FIG. 7B may be at least 50%, 60%, 70%, 80%, 90%, 95%, or even98%. Such portions of nucleic acids may be used, for instance, asprimers and probes for research and diagnostic purposes. Portions ofnucleic acids may be selected from any area of the sequences shown inFIG. 6B or FIG. 7B, for instance the first, second, third, etc., groupof 100 nucleic acids as numbered in the figures.

Recombinant nucleic acids, as mentioned above, may, for instance,contain all or portion of a disclosed nucleic acid operably linked toanother nucleic acid element such as a promoter, for instance, as partof a clone designed to express a protein. Cloning and expression systemsare commercially available for such purposes.

Various yeast strains and yeast-derived vectors are commonly used forexpressing and purifying proteins, for example, Pichia pastorisexpression systems are available from Invitrogen (Carlsbad, Calif.).Such systems include suitable Pichia pastoris strains, vectors,reagents, sequencing primers, and media. A similar system for expressionof proteins in Saccharomyces cerevisiae is also available fromInvitrogen, which includes vectors, reagents and media. For example, anucleotide sequence (e.g., a gene coding for the Δ⁵- or Δ⁸-desaturaseenzyme of the invention) may be cloned into the yeast expression vectorpYES2 and expressed under the control of an inducible promoter, such asa galactose-inducible promoter (GAL1).

Non-yeast eukaryotic vectors may also be used for expression of thedesaturases of the invention. Examples of such systems are the wellknown Baculovirus system, the Ecdysone-inducible mammalian expressionsystem that uses regulatory elements from Drosophila melanogaster toallow control of gene expression, and the Sindbis viral expressionsystem that allows high level expression in a variety of mammalian celllines. These expression systems are also available from Invitrogen.

Standard prokaryotic cloning vectors may also be used, for examplepBR322, pUC18 or pUC19 as described in Sambrook et al, 1989. Nucleicacids encoding the desaturases of the invention may be cloned into suchvectors that may then be transformed into bacteria such as Escherichiacoli (E. coli) which may then be cultured so as to express the proteinof interest. Other prokaryotic expression systems include, for instance,the arabinose-induced pBAD expression system that allows tightlycontrolled regulation of expression, the IPTG-induced pRSET system thatfacilitates rapid purification of recombinant proteins and theIPTG-induced pSE402 system that has been constructed for optimaltranslation of eukaryotic genes. These three systems are availablecommercially from Invitrogen and, when used according to themanufacturer's instructions, allow routine expression and purificationof proteins.

Alternatively, and of particular importance to this invention, a plantexpression system could be used. Plant expression systems arecommercially available. A gene of interest of the invention may becloned into a vector and the construct used to transform a plant cell.Any well known vector suitable for stable transformation of plant cellsand/or for the establishment of transgenic plants may be used, includingthose described in, e.g., Pouwels et al., Cloning Vectors: A LaboratoryManual, 1985, supp. 1987; Weissbach and Weissbach, Methods for PlantMolecular Biology, Academic Press, 1989; and Gelvin et al., PlantMolecular Biology Manual, Kluwer Academic Publishers, 1990. Such plantexpression vectors can include expression control sequences (e.g.,inducible or constitutive, environmentally or developmentally regulated,or cell- or tissue-specific expression-control sequences).

Examples of constitutive plant promoters useful for expressingdesaturase enzymes in plants include, but are not limited to, thecauliflower mosaic virus (CaMV) 35S promoter (see, e.g., Odel et al.,Nature 313:810, 1985; Dekeyser et al., Plant Cell 2:591, 1990; andTerada and Shimamoto, Mol. Gen. Genet. 220:389, 1990); the nopalinesynthase promoter (An et al., Plant Physiol. 88:547, 1988) and theoctopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989).

A variety of plant-gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for protein expression in plant cells, including promotersregulated by (1) heat (Callis et al., Plant Physiol. 88:965, 1988), (2)light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471,1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell 3:997, 1991;or chlorophyll a/b-binding protein promoter, Simpson et al., EMBO J.4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al.,Plant Cell 1:969, 1989), (4) wounding (e.g., wunI, Siebertz et al.,Plant Cell 1:961, 1989); or (5) chemicals such as methyl jasmonate,salicylic acid, or a safener. It may also be advantageous to employ (6)organ-specific promoters (e.g., Roshal et al., EMBO J. 6:1155, 1987;Schernthaner et al., EMBO J. 7:1249, 1988; Bustos et al., Plant Cell1:839, 1989; Zheng et al., Plant J. 4:357-366, 1993). Tissue-specificexpression may be facilitated by use of certain types of promoters, forexample, the napin promoter is a seed-storage protein promoter fromBrassica and specific to developing seeds. The β-conglycinin promotersdrive the expression of recombinant nucleic acids thus allowing, the Δ⁵or Δ⁸ proteins of the invention to be expressed only in specifictissues, for example, seed tissues.

Plant expression vectors can include regulatory sequences from the3′-untranslated region of plant genes (Thornburg et al., Proc. Natl.Acad. Sci. USA 84:744, 1987; An et al., Plant Cell 1:115, 1989), e.g., a3′ terminator region to increase mRNA stability of the mRNA, such as thePI-II terminator region of potato or the octopine or nopaline synthase3′ terminator regions.

Useful dominant selectable marker genes for expression in plant cellsinclude, but are not limited to: genes encoding antibiotic-resistancegenes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418,streptomycin, or spectinomycin); and herbicide-resistance genes (e.g.,phosphinothricin acetyltransferase). Useful screenable markers include,but are not limited to, β-glucuronidase and green fluorescent protein.

The invention also provides cells or plants or organisms transformedwith recombinant nucleic acid constructs that include all or a portionof the newly discovered polynucleotides that encode the novel Δ⁵ and/orΔ⁸ desaturase enzymes. An example of such a transformed plant ororganism would be a potato, tomato, rapeseed, sunflower, soy, wheat, orcorn plant. Multi-celled fungi, such as edible mushrooms, may also betransformed. Transformed oil-seed plants are of particular interest as20-carbon polyunsaturated fatty acids would accumulate within theseed-oil.

Nucleic acid constructs that express a nucleic acid according to theinvention can be introduced into a variety of host cells or organisms inorder to alter fatty acid biosynthesis. Higher plant cells, eukaryotic,and prokaryotic host cells all may be so transformed using anappropriate expression system as described above.

After a cDNA (or gene) encoding a desaturase has been isolated, standardtechniques may be used to express the cDNA in transgenic plants in orderto modify the particular plant characteristic. The basic approach is toclone the cDNA into a transformation vector, such that the cDNA isoperably linked to control sequences (e.g., a promoter) directingexpression of the cDNA in plant cells. The transformation vector is thenintroduced into plant cells by any of various techniques, for example byAgrobacterium-mediated transformation of plants or plant tissues, or byelectroporation of protoplasts, and progeny plants containing theintroduced cDNA are selected. All or part of the transformation vectorstably integrates into the genome of the plant cell. That part of thetransformation vector that integrates into the plant cell and thatcontains the introduced cDNA and associated sequences for controllingexpression (the introduced “transgene”) may be referred to as the“recombinant expression cassette.”

Selection of progeny plants containing the introduced transgene may bemade based upon the detection of an altered phenotype. Such a phenotypemay result directly from the cDNA cloned into the transformation vectoror may be manifested as enhanced resistance to a chemical agent (such asan antibiotic) as a result of the inclusion of a dominant selectablemarker gene incorporated into the transformation vector.

Successful examples of the modification of plant characteristics bytransformation with cloned cDNA sequences are replete in the technicaland scientific literature. Selected examples, which serve to illustratethe knowledge in this field of technology include:

U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”)

U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”)

U.S. Pat. No. 5,510,471 (“Chimeric Gene for the Transformation ofPlants”)

U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic Plants”)

U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for DiseaseResistance”)

U.S. Pat. No. 5,589,615 (“Process for the Production of TransgenicPlants with Increased Nutritional Value Via the Expression of Modified2S Storage Albumins”)

U.S. Pat. No. 5,750,871 (“Transformation and Foreign Gene Expression inBrassica Species”)

U.S. Pat. No. 5,268,526 (“Overexpression of Phytochrome in TransgenicPlants”)

U.S. Pat. No. 5,262,316 (“Genetically Transformed Pepper Plants andMethods for their Production”)

U.S. Pat. No. 5,569,831 (“Transgenic Tomato Plants with AlteredPolygalacturonase Isoforms”)

These examples include descriptions of transformation vector selection,transformation techniques, and the construction of constructs designedto over-express the introduced cDNA. In light of the foregoing and theprovision herein of the desaturase amino acid sequences and nucleic acidsequences, it is thus apparent that one of skill in the art will be ableto introduce the cDNAs, or homologous or derivative forms of thesemolecules, into plants in order to produce plants having enhanceddesaturase activity. Furthermore, the expression of one or moredesaturases in plants may give rise to plants having increasedproduction of poly-unsaturated fatty acids.

The invention also pertains to antibodies to the desaturase enzymes, andfragments thereof, these antibodies may be useful for purifying anddetecting the desaturases. The provision of the desaturase sequencesallows for the production of specific antibody-based binding agents tothese enzymes.

Monoclonal or polyclonal antibodies may be produced to the desaturases,portions of the desaturases, or variants thereof. Optimally, antibodiesraised against epitopes on these antigens will specifically detect theenzyme. That is, antibodies raised against the desaturases wouldrecognize and bind the desaturases, and would not substantiallyrecognize or bind to other proteins. The determination that an antibodyspecifically binds to an antigen is made by any one of a number ofstandard immunoassay methods; for instance, Western blotting, Sambrooket al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., Vols.1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989.

To determine that a given antibody preparation (such as a preparationproduced in a mouse against the Δ⁵-desaturase) specifically detects thedesaturase by Western blotting, total cellular protein is extracted fromcells and electrophoresed on a SDS-polyacrylamide gel. The proteins arethen transferred to a membrane (for example, nitrocellulose) by Westernblotting, and the antibody preparation is incubated with the membrane.After washing the membrane to remove non-specifically bound antibodies,the presence of specifically bound antibodies is detected by the use ofan anti-mouse antibody conjugated to an enzyme such as alkalinephosphatase; application of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium results in the production of a densely blue-coloredcompound by immuno-localized alkaline phosphatase.

Antibodies that specifically detect a desaturase will, by thistechnique, be shown to bind substantially only the desaturase band(having a position on the gel determined by the molecular weight of thedesaturase). Non-specific binding of the antibody to other proteins mayoccur and may be detectable as a weaker signal on the Western blot(which can be quantified by automated radiography). The non-specificnature of this binding will be recognized by one skilled in the art bythe weak signal obtained on the Western blot relative to the strongprimary signal arising from the specific anti-desaturase binding.

Antibodies that specifically bind to desaturases belong to a class ofmolecules that are referred to herein as “specific binding agents.”Specific binding agents that are capable of specifically binding to thedesaturase of the present invention may include polyclonal antibodies,monoclonal antibodies, and fragments of monoclonal antibodies such asFab, F(ab′)2, and Fv fragments, as well as any other agent capable ofspecifically binding to one or more epitopes on the proteins.

Substantially pure desaturase suitable for use as an immunogen can beisolated from transfected cells, transformed cells, or from wild-typecells. Concentration of protein in the final preparation is adjusted,for example, by concentration on an Amicon filter device, to the levelof a few micrograms per milliliter. Alternatively, peptide fragments ofa desaturase may be utilized as immunogens. Such fragments may bechemically synthesized using standard methods, or may be obtained bycleavage of the whole desaturase enzyme followed by purification of thedesired peptide fragments. Peptides as short as three or four aminoacids in length are immunogenic when presented to an immune system inthe context of a Major Histocompatibility complex (MHC) molecule, suchas MHC class I or MHC class II. Accordingly, peptides comprising atleast 3 and preferably at least 4, 5, 6, or more consecutive amino acidsof the disclosed desaturase amino acid sequences may be employed asimmunogens for producing antibodies.

Because naturally occurring epitopes on proteins frequently compriseamino acid residues that are not adjacently arranged in the peptide whenthe peptide sequence is viewed as a linear molecule, it may beadvantageous to utilize longer peptide fragments from the desaturaseamino acid sequences for producing antibodies. Thus, for example,peptides that comprise at least 10, 15, 20, 25, or 30 consecutive aminoacid residues of the amino acid sequence may be employed. Monoclonal orpolyclonal antibodies to the intact desaturase, or peptide fragmentsthereof may be prepared as described below.

Monoclonal antibody to any of various epitopes of the desaturase enzymesthat are identified and isolated as described herein can be preparedfrom murine hybridomas according to the classic method of Kohler &Milstein, Nature 256:495, 1975, or a derivative method thereof. Briefly,a mouse is repetitively inoculated with a few micrograms of the selectedprotein over a period of a few weeks. The mouse is then sacrificed, andthe antibody-producing cells of the spleen isolated. The spleen cellsare fused by means of polyethylene glycol with mouse myeloma cells, andthe excess unfused cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare diluted and aliquots of the dilution placed in wells of a microtiterplate where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall (Enzymol. 70:419, 1980), or a derivative methodthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988.

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein, which can be unmodified or modified, to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Forexample, small molecules tend to be less immunogenic than othermolecules and may require the use of carriers and an adjuvant. Also,host animals vary in response to site of inoculations and dose, withboth inadequate and excessive doses of antigen resulting in low-titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al., J. Clin.Endocrinol. Metab. 33:988-991, 1971.

Booster injections can be given at regular intervals, and antiserumharvested when the antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al., Handbook of Experimental Immunology, Wier,D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/mL of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves using conventional methods.

Antibodies may be raised against the desaturases of the presentinvention by subcutaneous injection of a DNA vector that expresses theenzymes in laboratory animals, such as mice. Delivery of the recombinantvector into the animals may be achieved using a hand-held form of theBiolistic system (Sanford et al., Particulate Sci. Technol. 5:27-37,1987, as described by Tang et al., Nature (London) 356:153-154, 1992).Expression vectors suitable for this purpose may include those thatexpress the cDNA of the enzyme under the transcriptional control ofeither the human β-actin promoter or the cytomegalovirus (CMV) promoter.Methods of administering naked DNA to animals in a manner resulting inexpression of the DNA in the body of the animal are well known and aredescribed, for example, in U.S. Pat. No. 5,620,896 (“DNA VaccinesAgainst Rotavirus Infections”); U.S. Pat. No. 5,643,578 (“Immunizationby Inoculation of DNA Transcription Unit”); and U.S. Pat. No. 5,593,972(“Genetic Immunization”), and references cited therein.

Antibody fragments may be used in place of whole antibodies and may bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz, Methods Enzymol. 178:476-496, 1989;Glockshuber et al. Biochemistry 29:1362-1367, 1990; and U.S. Pat. No.5,648,237 (“Expression of Functional Antibody Fragments”); U.S. Pat. No.4,946,778 (“Single Polypeptide Chain Binding Molecules”); and U.S. Pat.No. 5,455,030 (“Immunotherapy Using Single Chain Polypeptide BindingMolecules”), and references cited therein.

EXPERIMENTAL EXAMPLES Example 1 Organism Strains and Culture

The strain Euglena gracilis Z was obtained from Columbia Scientific. Theorganism was cultured on Cramer and Meyers medium (Cramer, and Meyers,Archiv fur Mikrobiologie 17:384-402, 1952) with the addition of sucroseas a carbon source. Cultures were maintained at 25° C. in absolutedarkness.

C. elegans was obtained from Caenorhabditis Genetics Center, St. Paul,Minn., and grown under standard conditions (Sulston et al., The NematodeCaenorhabditis elegans (Wood, W. B., Eds.), pp. 587-606, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

Example 2 Database Searches for C. elegans Gene Homologs

The Sanger Center(http://www.sanger.ac.uk/projects/c_elegans/blast_server.shtml) C.elegans genomic database was searched using BLAST™ with sequences ofplant desaturase enzymes, including the B. officinalis Δ ⁶-desaturase(GenBank accession number U79010). Two C. elegans polypeptides with thehighest scores were a peptide on cosmid W08D2 (high score 163), and oneon T13F2 (high score 121).

Example 3 RNA Isolation, Reverse Transcription PCR, and RACE (RapidAmplification of cDNA Ends)

For the E. gracilis Δ ⁸ gene, total RNA was isolated from heterotrophiccultures of E. gracilis using a phenol-SDS protocol (Ausubel, CurrentProtocols In Molecular Biology, 1988). Messenger RNA was purified fromtotal RNA using the PolyA-tract system (Promega Scientific, Madison,Wis.). Reverse transcription reactions were carried out usingSuperscript II (Life Technologies, Rockville, Md.). First-strandsynthesis in the initial reactions was primed using anchored polyTprimers (Clontech, Palo Alto, Calif.). Second-strand synthesis wasconducted as described (Life Technologies), and polymerase chainreaction amplification of the core region of the gene was accomplishedusing the primers (GGCTGGCTGACNCAYGARTTYTGYCAY; SEQ. ID NO. 5) and(CATCGTTGGAAANARRTGRTGYTCDATYTG; SEQ. ID NO. 6), designed to becompletely degenerate to sequences overlapping the first and thirdHis-box regions of the Δ⁶-desaturase C. elegans gene.

The amplification protocol was developed using published guidelines foruse of degenerate primers (Compton, PCR Protocols: A Guide To MethodsAnd Applications, 1990). The amplification consisted of 5 preliminarycycles at very low annealing temperature (30 seconds at 94° C., 1-minuteramp to 37° C., 45 seconds at 37° C., 3-minute ramp to 72° C.) followedby 30 cycles with higher temperature (30 seconds at 94° C., 1-minuteramp to 50° C., 45 seconds at 50° C., 3-minute ramp to 72° C.Preliminary amplifications to optimize thermal cycling parameters usedPfu DNA polymerase (Stratagene, La Jolla, Calif.). Amplification wassuccessful at 3 mM magnesium and each primer at 4 μM. Subsequently Taqpolymerase was used for amplification under identical conditions.

Polymerase chain reaction products from 350 to 750 bp were isolated fromagarose gels with commercial reagents (Qiagen, Valencia, Calif.) andsequenced directly using the degenerate primers and dye-terminationsequencing technology (Applied Biosystems, Foster City, Calif.). A groupof identical amplification products contained an open reading frame thatwas homologous to known desaturases when analyzed by BLAST™ search(Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997). The 5′ and 3′sequences of the complete mRNA were obtained with the Marathon RACEsystem (Clontech), using pairs of nested primers designed to amplifyfrom within the core sequence. To clone the complete 5′ end of the gene,it was necessary to repeat the reverse transcription with a primerspecific to the sequence of the open reading frame and repeat the 5′RACE amplification.

For the C. elegans Δ ⁵ gene, RNA isolation and reverse transcription-PCRwere performed as follows. RT-PCR was used to amplify the codingsequences of the two putative desaturase genes. Total RNA from mixedstage C. elegans was used for the RT-PCR template. The nematodes weregrown on agar plates as described and RNA was isolated using thephenol/SDS method (Sluder et al., Dev. Biol. 184:303-319, 1997). RT-PCRwas performed using the Superscript™ One-Step RT-PCR system(Gibco-BRL/Life Technologies). Approximately 1 μg of total RNA was addedto a reaction mixture consisting 0.2 mM of each dNTP, 1.2 mM MgSO₄,Superscript II™ RT/Taq polymerase mix, and 200 μM of appropriatedownstream and upstream primers. The reactions were incubated at 50° C.for 30 min., then subject to 35 cycles of PCR amplification. For theT13F2.1 gene (fat-4) a 5′ primer corresponding to bases 34339-34361 ofcosmid T13F2 was used. SmaI, HindIII, and XhoI restriction sites wereadded to these sequences to facilitate cloning. The resulting primer(CCCGGGAAGCTTCTCGAGGAATTTTCAATCCTCCTTGGGTC; SEQ. ID NO: 7) anneals tothe cosmid T13F2 19-42 base pairs upstream of the putative start codonATG of the fat-4 gene. To amplify the 3′ end of the fat-4 gene, a primerwas used corresponding to the inverse complement of bases 37075-37095 ofcosmid T13F2, with the addition of SmaI and BamH1 sites to facilitatecloning the polynucleotide of interest:CCCGGGTGGATCCGGAACATATCACACGAAACAG; SEQ. ID NO. 8. This primer begins 93base pairs after the putative stop codon TAG and ends 20 base pairsupstream of the predicted polyadenylation signal (AAUAAA; SEQ ID NO: 9)of the fat-4 gene.

For the determination of trans-splicing of specific leader sequences,downstream primers corresponding to the complement of bases 35009-35028of the T13F2 (TCTGGGATCTCTGGTTCTTG; SEQ. ID NO: 10) were used for theT13F2.1 gene. The upstream PCR primers were either SL1-20 or SL2-20(Spieth et al., Cell 73:521-532, 1993). The C. elegans homologue of theribosomal-protein L37 was used as an SL1-specific control, and K06H7.3was used as an SL2-specific control (Zorio et al., Nature 372:270-272,1994. SL1-20, SL2-20, and control primers were kindly provided by DiegoA. R. Zorio. RT-PCR products visualized by gel electrophoresis wereconfirmed by blotting the gel and probing with gene-specificoligonucleotides corresponding to the appropriate gene as previouslydescribed (Spieth et al., Cell 73:21-532, 1993).

Example 4 PCR Amplification of the Genes Encoding Δ⁵ and Δ⁸ Desaturases

DNA and protein sequences were analyzed using the Wisconsin-GCG packageof programs (Devereux et al., Nucleic Acids Res. 12:387-95, 1984).

To clone the E. gracilis (Δ⁸) open reading frame as a single DNAfragment, a set of primers was used to prime a reverse transcriptionspecifically for the open reading frame. The primer for the 5′ end ofthe gene began 3 nucleotides before the start codon and included thefirst 26 nucleotides of the open reading frame. The 3′ primer wascomplementary to the sequence between 22 and 52 nucleotides downstreamfrom the predicted termination codon. This PCR amplification wasconducted with Pfu polymerase to minimize the chance of an amplificationerror. The PCR reactions produced a single band of the predicted sizewhen analyzed by agarose gel electrophoresis. This band was cloned intothe vector pCR-Script Cam™ (Stratagene), and a single clone designatedpJW541 was chosen for analysis.

The plasmid containing SEQ ID NO: 3, described herein as pJW541, wasdeposited in the American Type Culture Collection (10801 UniversityBoulevard, Manassas, Va. 20110) on May 20, 2003 (ATCC Accession No.PTA-5206).

To express the C. elegans Δ ⁵-desaturase, the fat-4 cDNA amplificationproduct (see Example 3) was digested with HindIII and BamH1 and ligatedto the yeast expression vector pYES2 (Invitrogen) cut with HindIII andBamH1. The resulting plasmid was named pYFAT4.

Example 5 Expression of Δ⁵ and Δ⁸-Desaturases

For E. gracilis, the cloned Δ⁸ gene was transferred to the yeastexpression vector pYES2 (Invitrogen, Carlsbad, Calif.) by standardcloning techniques (Ausubel, Current Protocols In Molecular Biology,1988) using enzymes obtained from New England Biolabs, Beverly, Mass.The resulting yeast expression construct containing the open readingframe under the control of a galactose-inducible promoter was designatedpYES2-541.

Saccharomyces cerevisiae strain INVSc1 (Invitrogen) was transformed withpYES2-541 and cultured using standard methods (Ausubel, CurrentProtocols In Molecular Biology, 1988). Liquid medium containing 2%galactose was supplemented with fatty acid soaps (NuCheck Prep, ElysianMinn.) at a final concentration of 0.2 mM. Tergitol (1%, NP40) was addedto the yeast cultures to enhance fatty acid uptake (Stukey et al., J.Biol. Chem. 264:16537-16544, 1989), except for cultures containing 20:1,where 5% DMSO was substituted. Yeast were incubated overnight at 28° C.,harvested by centrifugation, washed once with 1% Tergitol, once with0.5% Tergitol, and finally once with distilled water.

For the C. elegans Δ ⁵ gene, the constructs were transformed intoSaccharomyces cerevisiae strain INVSc1 using the S.c. EasyComptransformation kit (Invitrogen). For experiments with the FAT-4 peptide,transformed yeast were grown overnight in uracil-deficient mediacontaining 2% galactose, 0.2 mM fatty acid, and 1% NP-40. Under theseconditions the percentage of these supplemented fatty acids which wereincorporated into yeast lipids ranged from 14-28% of the total yeastfatty acids. For experiments in which 20:1Δ¹¹ was used as a substrate,the 1% NP-40 was replaced by 5% DMSO to achieve better incorporation ofthis fatty acid.

Example 6 Analysis of Fatty Acids Using Gas Chromatography and GC-MassSpectrometry

Extraction of lipids and preparation of fatty acid methyl esters wascarried out by standard methods (Miguel and Browse, J. Biol. Chem.267:1502-1509, 1992). Gas chromatography of the methyl esters wasconducted by established methods (Spychalla et al., Proc. Natl. Acad.Sci. USA 94:1142-1147, 1997). Fatty acid 4,4-Dimethyloxazoline (DMOX)derivatives of yeast lipid extracts were prepared by standard methods(Fay and Richli, J. Chromatogr. 541:89-98, 1991). GC-mass spectrometrywas conducted on a Hewlett-Packard 6890 series GC-MS fitted with a 30m×0.25 μm HP5MS column, operating at an ionization voltage of 70 eV witha scan range of 50-550 Da. Fatty acids and their derivatives wereidentified where possible by comparison with authentic standards(NuCheck Prep).

Example 7 Identification and Amplification of the Euglena Δ ⁸-DesaturaseGene

Messenger RNA isolated from heterotrophic cultures was used as templatefor reverse transcription followed by PCR amplification using degenerateprimers that spanned the first and third conserved histidine-richregions of microsomal desaturase proteins. The C. elegans Δ ⁶-desaturasegene, FAT-3, was used as the principal basis for primer design. Tocompensate for the high degeneracy necessary in the primer pair,amplification reactions began with five cycles of low-temperatureannealing and a long temperature ramp between the annealing andpolymerization steps. Preliminary amplifications to optimize thermalcycling parameters used the proofreading Pfu DNA polymerase. Aftersuccessful Pfu amplification reactions using high primer and magnesiumconcentrations, Taq polymerase was used to generate a number of bandsdetectable on agarose gels.

Several of these bands, of approximately 650 bp, had identical sequence.This DNA sequence contained an open reading frame in which the predictedamino acid sequence was homologous to other membrane desaturases, andincluded a characteristic central His-box. Primers designed to bespecific to the amplified sequence were used to amplify the termini ofthe cDNA using 3′ and 5′ RACE techniques. The full-length cDNA for thisgene was 1745 bp in length. It included an open reading frame of 1272 bpand a 472-bp 3′ untranslated region. Most Euglena messenger RNAs areprocessed through the addition of a short 5′ RNA leader sequence, thetrans-spliced leader (Tessier et al., Embo. J. 10:2621-2625, 1991). ThisRNA processing step left a conserved sequence (TTTTTTTCG; SEQ. ID NO.11) at the beginning of each message (Cui et al., J. Biochem. (Tokyo)115:98-107, 1994). The presence of this leader in the cDNA sequenceconfirmed that the message was full-length at the 5′ end. RT-PCR withprimers flanking the open reading frame on the 5′ and 3′ ends resultedin a single band that was cloned into the vector pCR-Script Cam™(Stratagene), and designated pJW541. The gene corresponding to this ORFwas designated EFD1 (Euglena fatty acid desaturase 1).

Example 8 Similarity Between Euglena Δ ⁸-Desaturase and Other Proteins

The translated open reading frame indicated a protein of 422 amino acidswith a predicted molecular mass of 48.8 kDa. (FIG. 3). A BLAST™ searchof sequence databases revealed that the predicted protein sequenceexhibited regions of homology with the known group of membrane fattyacid desaturases, especially in the highly conserved histidine-richregions (Shanklin et al., Biochemistry 33:12787-12794, 1994).

Each of the His-box motifs is present in the EFD1 protein. The first(HXXXH; SEQ ID NO: 17) starts at amino acid 146 and the second (HXXHH;SEQ ID NO: 12) at amino acid 183 (FIG. 3). EFD1 contains a variant thirdHis-box, QXXHH (SEQ ID NO: 13), starting at amino acid 361, similar tothe cloned Δ⁵- and Δ⁶-desaturases. EFD1 exhibits conservation of proteinsequence in the regions surrounding the highly conserved regions,especially with FAT-3 and FAT-4, the Δ⁶- and Δ⁵-desaturases of C.elegans (FIG. 3). Outside the highly conserved regions, the amino acidsequence shows considerably less similarity to other desaturases.Overall, the amino acid identity with FAT-3 and FAT-4 is 33%, comparedto 28% identity with the borage Δ⁶-desaturase.

EFD1 also contains a cytochrome b₅-like motif at its N-terminus. Theprotein encodes seven of the eight most highly conserved amino acidscharacteristic of cytochrome b₅ (FIG. 3), which are responsible for hemebinding. Similar motifs are found at the N-terminal regions of FAT-3 andFAT-4 (FIG. 3), and the borage Δ⁶ protein, as well as the carboxylterminus of a yeast Δ⁹ protein.

The structure of the Euglena protein also exhibits similarities withknown desaturases. Membrane desaturases are type II multiple membranespanning proteins, and hydropathy analysis of the cloned Euglena geneindicates that the predicted protein has at least three significanthydrophobic regions long enough to span the membrane bilayer twice. Asis true for most desaturase enzymes, there are 31 amino acid residuesbetween the first two His-boxes. The distance between the between thesecond and third His-box is 173 residues, within the range previouslyobserved (Shanklin and Cahoon, Annu. Rev. Plant Physiol. Plant Mol.Biol. 48:611-641, 1998).

Example 9 Activity of the Euglena Δ ⁸-Desaturase Protein

To confirm the activity of the enzyme, the EFD1 cDNA was transferredfrom pJW541 to yeast expression vector pYES2 under the control of agalactose-inducible promoter. The resulting construct, pYES2-541, wasintroduced into S. cerevisiae. Yeast membranes do not contain 20-carbonfatty acids but incorporate them from the culture medium. Accordingly,yeast cultures were supplemented with various fatty acid soaps, using ayeast strain containing the empty vector as control, and analyzed thefatty acids of the cultures by methyl-ester derivatization and gaschromatography.

The patterns of desaturation activity in these experiments indicatedthat pYES2-541 expresses a Δ⁸-desaturase enzyme that does not have Δ⁵ orΔ⁶ activity. The ability of the experimental yeast strain to produce Δ⁸desaturation was shown when the culture medium was supplemented with20:2 (FIG. 4). A desaturation peak whose retention time is identical toauthentic 20:3 was produced. The vector-only control culture did notdesaturate 20:2 (FIG. 4). The yeast strain expressing the Euglena genealso desaturated 20:3 and 20:1 (FIG. 4), again without desaturationactivity in the control cultures. The cloned Euglena protein was mostactive with 20:3 and 20:2 as substrates, desaturating 70% and 73% of thetotal incorporated 20-carbon fatty acid. EFD1 was least active with20:1, converting 32% of that substrate to a desaturation product (FIG.4).

When the culture medium was supplemented with a substrate forΔ⁵-desaturation, 20:4, the fatty acid was incorporated into the yeast,but no 20:5 desaturation product was produced. Similarly, when themedium was supplemented with 18:2 and 18:3, no desaturation occurred,demonstrating that the cloned gene did not express a Δ⁶-desaturase.

To confirm that desaturation had occurred at the Δ⁸ position,4,4-dimethyloxazoline (DMOX) derivatives of yeast fatty acids wereanalyzed by GC-MS. DMOX derivatives have mass spectra that are moreeasily interpreted than spectra of methyl esters, and permit unambiguousdetermination of double-bond locations in polyunsaturated fatty acids(Christie, Lipids 33:343-353, 1998). For the experiment that desaturatedthe fatty acid 20:2, the retention time of the product on the GC-MSinstrument was 16.8 min., identical to DMOX-derivatized authentic 20:3.The mass spectrum of this desaturation product and its molecular ion(m/z 359) indicated that it was the 20:3 compound. Two spectralfrequency peaks at m/z 182 and 194, separated by only 12 a.m.u., showedthat the introduced double bond was at the Δ⁸ position (FIG. 5). (Thesubstrate 20:2, which is saturated at the 8-position, has peaks at 182and 196, separated by 14 a.m.u.) The spectrum of the product, with itsdesaturation peaks, was identical to that of authentic 20:3 (Luthria andSprecher, Lipids 28:561-564, 1993). The other substrates, 20:1 and 20:3,were also desaturated at the Δ⁸-position by EFD1. The peaks at m/z 182and m/z 194 appeared in the spectrum of each, and the molecular ion wasreduced from that of the substrate by two in each case (FIG. 5).

Example 10 Identification and Cloning of Two Fatty Acid C. elegansDesaturase Genes

Two high-scoring open reading frames were identified during a search ofthe C. elegans genomic DNA database with the borage Δ⁶-desaturaseprotein sequence. Both proteins predicted from these open readingframes, W08D2.4 and T13F2.1, contained an N-terminal sequence resemblingcytochrome b₅, including the characteristic (HPGG) heme binding domain,and an H→Q substitution in the third histidine box. The W08D2.4 gene wasdenoted fat-3 and the T13F2.1 gene was denoted fat-4, since they bothappear to encode fatty acid desaturases. Interestingly, the fat-3 andfat-4 genes are located next to each other on overlapping cosmids in thesame 5′ to 3′ orientation, with only 858 nucleotide base pairsseparating the putative polyadenylation signal of the fat-4 gene and theATG start codon of the fat-3 gene (FIG. 8). This gene organization isreminiscent of operons, in which two or more genes are transcribed underthe control of a single promoter and regulatory region.

In C. elegans the polycistronic pre-mRNA is converted to monocistronicmRNA by cleavage and polyadenylation at the 3′ end of the upstream geneand transplicing to the SL2 sequence at the 5′ end of the downstreamgene, with the two mRNAs being subsequently independently translated.However, out of more than 30 such operons that have been analyzed, thedistances between the 3′ end of the upstream gene and the 5′ end of thedownstream gene are generally about 100 base pairs, with a few separatedby 300-400 base pairs (Blumenthal et al, C. elegans II, pp. 117-145,Cold Spring Harbor Laboratory Press, Cold Spring, N.Y., 1997).

The C. elegans fat-3 and fat-4 genes were tested to determine whetherthey were trans-spliced to either SL1 or SL2 in order to determine ifthey might be co-transcribed in a single operon. It was found that thefat-4 gene was transpliced to SL1, but that the fat-3 gene was nottranspliced to either spliced leader sequence. Therefore, it wasconcluded that each gene contains its own 5′ promoter and regulatoryregion.

Both of these genes were cloned using RT-PCR. The fat-4 gene sequencematched the T13F2 genomic sequence exactly. However the gene productencoded by the cDNA was seven amino acids shorter than predicted byGenefinder for T13F2.1 (GenBank accession number Z81122) because the DNAsequence encoding amino acid residues 198-204 was not present in thefat-4 cDNA. The resulting peptide length was 447 amino acids instead ofthe previously predicted 454 amino acids. The gene product encoded bythe fat-3 cDNA also matched the genomic sequence of W08D2.4 (GenBankaccession number Z70271) perfectly. However, the gene product was alsoshorter than the predicted protein sequence. Codons for amino acidresidues 38-67 of W08D2.4 were not present in the cDNA. In both casesthe gene-prediction software used in the genomic sequencing projectappeared to have misidentified some intron DNA as coding sequences.

Example 11 Sequence Comparisons for C. elegans Δ ⁵

The C. elegans FAT-3 and FAT-4 proteins, the Mortierella alpina Δ⁵-desaturase, and the B. officinalis Δ ⁶-desaturase appear to beproteins of similar structure in that they all contain an N-terminalcytochrome b₅ domain, three histidine boxes, and distinct hydrophobicmembrane-spanning domains predicted by the TMHMM program from the Centerfor Biological Sequence Analysis, Technical University of Denmark(http://www.cbs.dtu.dk/services/TMHMM-1.0/). The predicted structure isconsistent with the proposed desaturase structural model (Stukey et al.,J. Biol. Chem., 265:20144-20149, 1990). Despite these similarities, theoverall sequence identity among the four proteins is quite low. Forexample, the FAT-3 Δ⁶-desaturase and the borage Δ⁶-desaturase share only28% identity on the amino acid level. The fat-4 gene product shares 25%amino acid identity with the borage Δ⁶-desaturase and 19% amino acididentity with the Mortierella alpina Δ ⁵-desaturase. Indeed the onlyportion of the FAT-4 protein that shows extended homology to the M.alpina Δ ⁵-desaturase is a sequence of 36 residues incorporating thethird His box which has 44% identity and 56% similarity. The mostclosely related pair of sequences are fat-3 and fat-4, which are 46%identical on the amino acid level and 54% identical over the entire cDNAsequence.

FIG. 9 shows the sequence comparison of the borage Δ⁶-desaturase, C.elegans FAT-3, C. elegans FAT-4, and the Mortierella alpina Δ⁵-desaturase. The similar heme-binding domains (HPGG) and the threehistidine box regions are underlined. The presence of these conservedmotifs indicate that the fat-4 gene may encode a desaturase or a relatedfatty acid modifying enzyme. However it is not possible, from thesesequence comparisons alone, to predict whether this gene encodes aΔ⁶-desaturase, a Δ⁵-desaturase, or a more distantly related enzyme.

Example 12 Fatty Acid Desaturase Activity and Substrate Specificity inYeast for C. elegans Δ ⁵

To determine the enzymatic activity of the FAT-4 desaturase-likeprotein, the protein was expressed in Saccharomyces cerevisiaesupplemented with polyunsaturated fatty acid substrates that are notnormally present in this yeast. The FAT-4 protein was expressed in theyeast expression vector pYES2 from the GAL1 promoter by growing thecells in the presence of galactose and various fatty acids. After 16hours of growth, the cells were analyzed for total fatty acidcomposition by gas chromatography (GC). Comparison of cells supplementedwith di-homo-γ-linolenic acid (20:3Δ^(8,11,14)) carrying pYES2containing the fat-4 coding sequence and cells carrying the vector alonerevealed the presence of a major new peak eluting at 14.49 minutes inthe cells expressing FAT-4 (FIG. 10B). The novel peak had a retentiontime identical to that of the authentic arachidonic acid methyl ester(20:4Δ^(5,8,11,14)), and was determined to be arachidonic acid(20:4Δ^(5,8,11,14)) because its mass spectrum was identical to that ofauthentic arachidonic acid methyl ester, including a mass ion peak atm/z 318.

The identity of this compound was further verified by converting theyeast fatty acid methyl esters into oxazoline derivatives in order toproduce structure specific mass spectra which simplify the determinationof double-bond positions in a hydrocarbon chain. The mass spectrum ofthe DMOX derivative of the novel 20:4 component was consistent with thepublished spectrum for arachadonic acid and contained a prominent peakat m/z 153, which is diagnostic of a double bond at the Δ⁵ position.Therefore, it was concluded that the fat-4 gene encodes a Δ⁵-desaturasecapable of synthesizing arachidonic acid from the substratedi-homo-γ-linolenic acid. In contrast, the FAT-4 protein showed noactivity when linoleic acid (18:2Δ^(9,12)) or γ-linolenic acid(18:3Δ^(9,12,15)) were provided as substrates, indicating an absence ofΔ⁶-desaturase activity.

Further analysis of the GC trace of the total fatty acids of the yeastcells expressing fat-4 revealed the presence of a second novel peakeluting at 12.91 minutes which was not present in the empty vectorcontrol cells. Analysis of the mass spectrum of this novel peak revealeda molecular ion species of 294, identical to that of a methyl ester ofan 18-carbon fatty acid with two double bonds (18:2), but its retentiontime and mass spectrum were not identical to the common isomer 18:2Δ^(9,12).

In microsomal extracts of mammalian liver, Δ⁵-desaturase activity hasbeen reported to act on a number of 18 and 20-carbon precursors toproduce uncommon fatty acids such as 18:2Δ^(5,11), 20:3Δ^(5,11,14) and20:4Δ^(5,11,14,17) (28, 29). Two species of slime molds have also beenreported to produce small amounts of 18:2Δ^(5,9), 18:2Δ^(5,11),20:3Δ^(5,11,14) and 20:4Δ^(5,11,14,17) (Rezanka, Phytochemistry,33:1441-1444, 1993). These fatty acids are unusual in that their doublebonds do not follow the conventional methylene-interrupted pattern (onedouble bond every three carbons).

Therefore, it was suspected that the novel peak exhibited on the GCspectrum is a result of the C. elegans Δ ⁵-desaturase acting on 18:1Δ⁹(or 18:1Δ¹¹, which in S. cerevisiae constitutes 15-20% of the total18:1) compound, to produce the uncommon isomer 18:2Δ^(5,9) or18:2Δ^(5,11). These yeast fatty acid methyl esters were converted intooxazoline derivatives. It was found that the mass spectrum of the DMOXderivative of the novel 18:2 component contained the Δ⁵-specific peak atm/z 153. However the larger ion peaks characteristic of double bonds atthe Δ⁹ or Δ¹¹ position were not detected due to the small amount of thismolecule present in the total yeast extracts.

To test if the C. elegans Δ ⁵-desaturase was capable of desaturatingother substrates to produce other uncommon, non-methylene-interruptedfatty acids, the yeast expressing the FAT-4-desaturase was supplementedwith unconventional Δ⁵-substrates such as 20:1Δ¹¹, 20:2Δ^(11,14), and20:3Δ^(11,14,17). No novel peaks were detected when the substrate20:1Δ¹¹ was fed to yeast. However, when 20:2Δ^(11,14) and20:3Δ^(11,14,17) were provided as substrates, novel peaks were detectedeluting at 14.62 minutes and 14.69 minutes, respectively (FIGS. 11A and11B, respectively). The mass-spectrum analysis of DMOX derivatives ofthese molecules yielded results consistent with published values for20:3Δ^(5,11,14) and 20:4Δ^(5,11,14,17), including a prominent ion peakof m/z 153 (which is diagnostic of double bonds at the Δ⁵ position). Itwas found, however, that these fatty acids were not produced to the sameextent as arachidonic acid (20:4Δ^(5,8,11,14)) (FIG. 12). In theseexperiments, 55% of exogenously fed di-homo-γ-linolenic acid(20:3Δ^(8,11,14)) was converted to arachidonic acid, while only 5%, 27%,and 26% of the 18:1, 20:3Δ^(11,14), and 20:2Δ^(11,14,17) substrates wereconverted (FIG. 12).

The fat-3 gene was expressed in the yeast expression vector pMK195containing the constitutive ADH promoter. The FAT-3 protein was able todesaturate linoleic acid (18:2Δ^(9,12)) into γ-linolenic acid(18:3Δ^(6,9,12)), in agreement with published results (Napier et al.,Biochem. J. 330:611-614, 1998). It was also found that FAT-3 was capableof desaturating α-linolenic acid (18:3Δ^(9,12,15)) to 18:4Δ^(6,9,12,15),a common reaction in animals. The FAT-3 protein showed no activity on20:1Δ¹¹, 20:2Δ^(11,14), 20:3Δ^(8,11,14), or 20:3Δ^(11,14,17). Therefore,the substrate specificities of the C. elegans Δ ⁵ and Δ⁶-desaturaseswere determined to be specific and non-overlapping.

Example 13 Discussion of the E. gracilis Δ ⁸-Desaturase

Desaturation at the Δ⁸-position has not been reported for any previouslycloned gene (Tocher et al., Prog. Lipid Res. 37:73-117, 1998).

The predicted EFD1 protein has 33% amino acid identity with both FAT-3and FAT-4, (FIG. 9), while its identity with the cloned borageΔ⁶-desaturase is 28%. The highest sequence conservation is found in theHis-box motifs which are critical for desaturase activity, most likelybecause they serve as the diiron-oxo component of the active site(Shanklin et al., Biochemistry 33:12787-12794, 1994). Sequenceconservation is also evident in the N-terminal cytochrome b₅-likedomain, where most of the essential residues of cytochrome b₅ (Lederer,Biochimie 76:674-692, 1994) that are retained in FAT-3 and FAT-4 arealso present in the EFD1 protein (FIG. 3).

Expression of the EFD1 gene in yeast was used to characterize itsactivity. Three different 20-carbon substrates with double bonds at theΔ¹¹-position were desaturated (FIG. 4), and analysis of the productsindicated that, for each one, desaturation had taken place at the Δ⁸position (FIG. 5). The cloned Euglena desaturase showed a clearpreference for the substrates of metabolic significance with greaterthan two-fold preference for 20:2 and 20:3 over 20:1 (Ulsamer et al., J.Cell Biol. 43:105-114, 1969). Even though EFD1 is quite similar to othermicrosomal desaturases, its activity was specific, as evidenced by itsinactivity on substrates for Δ⁵ and Δ⁶ desaturation (FIG. 12).

The 20-carbon substrates for Δ⁸ desaturation are available in abundancein heterotrophically grown E. gracilis (FIG. 2). These same substratesalso are available in mammals, since 20:2 and 20:3 are produced byelongation from 18:2 and 18:3, in competition with the typical Δ⁶desaturation (FIG. 1). Labeling experiments with rat liver homogenatesindicate that elongation of the 18-carbon fatty acids is five-fold morerapid than the competing desaturation (Pawlosky et al., J. Lipid Res.33:1711-1717, 1992).

Implicit in the current understanding of the Δ⁶ pathway of 20-carbonpolyunsaturated fatty acid biosynthesis is a reliance on alternatingdesaturation and elongation to control flux through the pathway. Whileelongation often appears to be non-specific, most desaturations arespecific both as to chain length of the substrate and to existingdesaturation pattern of the fatty acid (Heinz, Lipid Metabolism inPlants, pp. 33-89, 1993). However, data from experiments in mammaliantissue (Bernert and Sprecher, Biochim. Biophys. Acta. 398:354-363, 1975;Albert et al., Lipids 14:498-500, 1979) and with yeast expressing the C.elegans Δ ⁵-desaturase gene (FIG. 12), indicate that Δ⁵ enzymesdesaturate fatty acids having a double bond at the Δ¹¹-position but notat the Δ⁸-position, producing the non-methylene-interrupted 20:3 and20:4 compounds at significant rates. For the Δ⁸ pathway, Δ⁸ desaturationoccurs in competition with Δ⁵ activity on the substrates 20:2 and 20:3.In spite of this promiscuity of Δ⁵ enzymes, lipid profiles of mammaliantissue do not contain fatty acids with the Δ^(5,11) (Heinz, LipidMetabolism in Plants, pp. 33-89, 1993; and Ulsamer et al., J. Cell Biol.43:105-114, 1969) desaturation pattern, nor are they seen in Euglena.

One explanation may be that Δ⁸-desaturation of the common substratesoccurs very rapidly, while Δ⁵-desaturation proceeds more slowly, so thatlittle Δ^(5,11) product is formed. In support of this explanation, theEuglena Δ ⁸ appears to be a very active desaturase when expressed inyeast (FIG. 4), compared to similarly expressed Δ⁵-desaturase enzymes.The Euglena desaturase must be sufficiently active to account for allthe long chain polyunsaturates of rapidly growing Euglena cultures (FIG.2). In contrast, the observed rates of Δ⁸-desaturation in mammaliantissue are relatively slow. The highest apparent rate occurs incancerous tissues without Δ⁶ activity, where Δ⁸ desaturation permitsproduction of arachidonic acid at only 17% of the level of comparablenormal cells with Δ⁶ activity (Grammatikos et al., Br. J. Cancer70:219-227, 1994).

Alternatively, the lack of Δ^(5,11) unsaturated fatty acids in membranescould be explained if the Δ⁸ desaturase accepts these fatty acids fordesaturation. The convention that Δ⁸-desaturation precedes Δ⁵ activity(FIG. 1) is based on observations of desaturation reactions proceedingsequentially along the fatty acid hydrocarbon chain. The reverse orderof desaturation, with Δ⁵-saturation preceding Δ⁸-desaturation, has beenclaimed (Takagi, J. Chem. Bull. Japan 38:2055-2057, 1965) and rejected(Schlenk et al., Lipids 5:575-577, 1970) in mammalian liver, andproposed as a likely pathway based on experiments with deuteratedsubstrates in glioma cells (Cook et al., J. Lipid Res. 32:1265-1273,1991).

In Euglena the products of Δ⁸-desaturation, 20:3Δ^(8,11,14) and20:4Δ^(8,11,14,17) may be incorporated directly into membranes, orsubjected to desaturation at the Δ⁵-position to produce arachidonic andeicosapentaenoic acids (Hulanicka et al., J. Biol. Chem. 239:2778-2787,1964). Further elongation and desaturation leads to severalpolyunsaturated 22-carbon fatty acids (FIG. 2). In mammals, whether theproducts are derived from Δ⁸ or Δ⁶ activity, similar processes producemostly arachidonic, eicosapentaenoic, and docosahexaenoic acid (22:6)(Hwang, Fatty Acids in Foods and Their Health Implications, pp. 545-557,1992; Bernert and Sprecher, Biochim. Biophys. Acta 398:354-363, 1975;Lees and Korn, Biochemistry 5:1475-1481, 1966; Albert and Coniglio,Biochim. Biophys. Acta 489:390-396, 1977; Bardon et al., Cancer Lett.99:51-58, 1996; and Sprecher and Lee, Biochim. Biophys. Acta.388:113-125, 1975), although some 20:3 is metabolized directly to series1 eicosanoid metabolic regulators (Hwang, Fatty Acids in Foods and TheirHealth Implications, pp. 545-557, 1992).

It is interesting to note that the alternate pathway of Δ⁸-desaturationbegins with an elongation step. This elongation is the standard pathwayin Euglena, which produces substantial amounts of 20:2 (7.4%) and 20:3(1.4%) (FIG. 2). In mammalian tissue with little or no Δ⁶ activity(Grammatikos et al., Br. J. Cancer 70:219-227, 1994), this would be thefirst step by which the essential fatty acids 18:2 and 18:3 aremetabolized to their 20-carbon derivatives. Recently there has been anew emphasis on fatty acid chain elongation acting as a regulatory stepin fatty acid biosynthesis (Garcia et al., Lipids 25:211-215, 1990;Sprecher et al., Prostag. Leukot. Essent. Fatty Acids 52:99-101, 1995).Evidence that breast cancer cells may selectively elongate 18:3 inpreference to 18:2, and that Δ⁸-desaturation follows this elongation(Bardon et al., Cancer Lett. 99:51-58, 1996) implies thatΔ⁸-desaturation may play an important role in some cancer cells.

The identification and cloning of a Δ⁸-desaturase gene permitexaminations of the alternate pathway for biosynthesis of 20-carbonpolyunsaturated fatty acids and will give insight into the possiblemechanisms of Δ⁸-desaturation. In mammals, operation of this alternativepathway may be confined to specialized tissues, where the demand forpolyunsaturated fatty exceeds the supply provided through rate-limitingΔ⁶ desaturation. The pathway may be of greater significance where Δ⁶desaturation is reduced or absent. Since fatty acid desaturasemetabolism is perturbed in many cell lines, both transformed(Grammatikos et al., Ann. N.Y. Acad. Sci. 745:92-105, 1994) anduntransformed (Rosenthal, Prog. Lipid Res. 26:87-124, 1987), it may bethat Δ⁸ activity is only revealed in the absence of Δ⁶ activity.Alternatively, Δ⁸ activity may arise or increase with cell neoplasia.The isolation and examination of this Δ⁸ gene, and analysis of itssubstrate specificity, should facilitate the determination of the roleof Δ⁸ activity in both normal and cancerous mammalian tissue.

Example 14 Discussion of the C. elegans Δ ⁵-Desaturase

In this example, we describe a region of the C. elegans genome locatedat position 4.88 of chromosome IV is described that contains the Δ⁵- andΔ⁶-desaturase genes. The amino acid sequences encoded by the two genesare 46% identical to each other, and each contains an N-terminal hemebinding domain typical of the electron carrier cytochrome b₅ and threehistidine boxes. Both genes contain the consensus sequence of the thirdHis box (QXXHH; SEQ. ID NO. 13) that has so far been shown to be uniqueto the microsomal desaturases involved in double-bond insertion atcarbons below position 9.

Despite these similarities, these two microsomal desaturases showabsolutely non-overlapping substrate specificities. When overexpressedin the yeast Saccharomyces cerevisiae, the C. elegans Δ ⁶-desaturase(FAT-3) specifically acts on two 18-carbon substrates, linoleic andγ-linolenic acid, and always desaturates in a methylene-interruptedpattern (one double bond every three carbons). The mammalianΔ⁶-desaturase system has likewise been demonstrated to insert doublebonds strictly in a methylene-interrupted pattern and to have noactivity on 20-carbon substrates (Schmitz et al., Lipids 12:307-313,1997). The C. elegans Δ ⁵-desaturase (FAT-4), in contrast, acts on anumber of 20-carbon substrates, as well as on an endogenous 18:1 fattyacid of yeast, and is capable of inserting double bonds in anon-methylene interrupted pattern.

Non-methylene-interrupted fatty acids such as 20:2 Δ^(5,11), 20:3Δ^(5,11,14), 18:2 Δ^(5,11) have been detected in mammalian cells byfeeding ¹⁴C-labeled substrates to rats raised on a fat-deficient diet(Ulman et al., Biochem. Biophys. Acta 248:186-197, 1971). However, thesefatty acids are considered to be “dead end” metabolites, as they havenot been demonstrated to serve as precursors to signaling molecules suchas prostaglandins, nor are they detectable in tissue lipids of rats whoare not preconditioned on a fat-deficient diet. (We also did not detectthese fatty acids in C. elegans lipid extracts.)

In yeast expressing the C. elegans Δ ⁵-desaturase gene, the amount ofsubstrate converted was greatest for the metabolically significantsubstrate 20:3 Δ^(8,11,14) (FIG. 13). The amount of 20:2Δ^(11,14) and20:3Δ^(11,14,17) that was desaturated less than half the amount ofconventional substrate that was desaturated. This was consistent withthe rates of desaturation in microsomal extracts of mammalian liver,where the rate of conversion of labeled 20:2Δ^(11,14) to 20:3Δ^(5,11,14)is 41% of the rate of conversion of labeled 20:3Δ^(8,11,14) to20:4Δ^(5,8,11,14) (Bernet et al., Biochem. Biophys. Acta 398:354-313,1975).

The C. elegans fat-3 and fat-4 genes are present in a gene cluster inthe same 5′ to 3′ orientation. Yet, unlike other gene clusters of thissort in C. elegans, the downstream fat-3 gene is not transpliced to SL2,and therefore is unlikely to be co-transcribed with the upstream fat-4gene. The two genes could be located next to each other as a result ofan ancient gene-duplication event. The DNA sequences share 54% identityover the entire cDNA coding sequence; however the genes do not share anycommon intron/exon boundaries (FIG. 8).

This is the first disclosed sequence of a Δ⁵-desaturase gene from ananimal. The sequence of the C. elegans Δ ⁵-desaturase is quite distantfrom the bacterial and fungal Δ⁵-desaturases that have been reported,and this animal sequence should facilitate the search fordesaturase-encoding sequences from humans and other mammals. Both theΔ⁵- and Δ⁶-desaturases are important regulatory enzymes in humans. Theyparticipate in critical steps in the pathway to produce precursors forsynthesis of hormone-like eicosanoid molecules from the essentialdietary fatty acids, linoleic acid and α-linolenic acid. The activitiesof these desaturases have been shown to be under hormonal andnutritional control, but the mechanism of this control is still unknown.

Certain diseases, such as diabetes, result in low Δ⁵-desaturaseactivity, while HTC cells, isolated from an ascites tumor derived from asolid hepatoma, show increased Δ⁵-desaturase activity. The availabilityof mutational and reverse genetic tools and the expanding knowledge ofcellular and developmental biology in C. elegans make this an attractivesystem to study the roles of polyunsaturated fatty acids and theirmetabolic products in development, reproduction, and other cellularprocesses of animals.

Example 15 A Plant Cell Transformed with the Δ⁵ and Δ⁸-Desaturase Genesof the Invention

Using the methods described herein, Δ⁵- and Δ⁸-desaturases of theinvention may be cloned and expressed in plants to produce plants withenhanced amounts of 20-carbon polyunsaturated fatty acids. Such plantsprovide an inexpensive and convenient source of these important fattyacids in a readily harvestable and edible form.

For instance, the Δ⁵- and Δ⁸-desaturases of the invention can be clonedinto a common food crop, such as corn, wheat, potato, tomato, yams,apples, pears, or into oil-seed plants such as sunflower, rapeseed, soy,or peanut plants. The resulting plant would express the appropriateenzyme that would catalyze the formation of 20-carbon polyunsaturatedfatty acids. In the case of an oil-seed plant, the seed oil would be arich source of 20-carbon polyunsaturated fatty acids.

The Δ⁵- and Δ⁸-desaturase genes may be cloned and expressed eitherindividually, or together in a host plant cell. The correspondingdesaturases can be expressed using a variety of different controlsequences, such as promoters, enhancers, and 3′-termination sequences.These control sequences can be used to control the expression of eachdesaturase individually. For example, the Δ⁵-desaturase can be clonedsuch that it is under the control of a strong promoter, and theΔ⁸-desaturase can be cloned such that it is under the control of a weakpromoter, thus yielding a transgenic plant that expresses moreΔ⁵-desaturase than Δ⁸-desaturase. Furthermore, expression can becontrolled by operably linking one or more of the desaturase genes ofinterest to a promoter that is activated by exposure of the plant cellto an appropriate regulatory agent such as an inducer, repressor,de-repressor or inhibitor agent. Such regulation is discussed above.Alternatively, expression of non-contiguous genes may be coordinated bylinking the expression of a first gene with the expression of an induceror de-repressor molecule that induces or de-represses the expression ofa second gene.

The genes of the invention can be integrated into the genome of a plant(for example, by Agrobacterium-mediated T-DNA transfer) or animal (forexample, by use of broad host-range retroviruses, e.g., an adenovirusvector) so that the Δ⁵ and Δ⁸-desaturases of the invention are expressedas part of the genome. For transgenic plants, the T-DNA vector may beused that would result in integration of transgenes (Δ⁵ and/or Δ⁸) intothe host cell genome.

For example, the expression of the Δ⁵- and Δ⁸-desaturases in a plant,such as Arabidopsis, can be achieved by constructing a planttransformation vector to introduce the cDNA of each desaturase into theplant. The vector can contain a tissue specific promoter so that thedesaturase protein will be expressed during seed development. Examplesof seed specific promoters include that for phaseolin (van der Geest andHall, Plant Mol. Biol. 32:579-88, 1996) or the promoter for napin(Stalberg et al., Plant Mol. Biol. 23: 671-83, 1993). Otherseed-specific promoters that can be used are those located on thegenomic BAC clone T24A18 (LOCUS ATT24A18. (1999) 45980 bp Arabidopsisthaliana DNA chromosome 4, ACCESSION #AL035680, NID g4490701) of theArabidopsis genome. These promoters regulate seed storage proteinexpression in Arabidopsis. Other promoters which express genesspecifically in seeds like those described in (Parcy et al., Plant Cell6:1567-1582, 1994) can also be used. The constructs containing thedesaturase coding sequence and promoter sequence can then transferred tostandard plant transformation T-DNA vectors similar to pART27 (Gleave,Plant Mol. Biol. 20:1203-1207, 1992), pGPTV (Becker et al., Plant Mol.Biol. 20:1195-7, 1992), or pJIT119 (Guerineau et al., Plant Mol. Biol.15:127-136, 1992). If the plant is to be transformed with twoconstructs, i.e. one encoding the Δ⁸-desaturase and the other encodingthe Δ⁵-desaturase, then it is preferable to choose two differentselectable markers so that only double transformants will regenerate.For example, the vector carrying the Δ⁵-desaturase can be constructedsuch that it contains the kanamycin (nptII) gene, and the vectorcarrying the Δ⁸-desaturase can be constructed such that it contains thephosphinothricin (bar) gene. Transformants are then selected on mediacontaining kanamycin and phosphinothricin. Transformation of Arabidopsisis readily achieved using the Agrobacterium-mediated vacuum infiltrationprocess (Katavic et al., Mol. Gen. Genet. 245:363-70, 1994) or thefloral dip modification of it (Clough and Bent, Plant J. 16:735-43,1998), although several other methods are also commonly used. Transgenicprogeny will be identified by selection using the appropriate antibioticor herbicide, either kanamycin or phosphinothricin, or both. Since theΔ⁸ and Δ⁵ constructs use different selectable markers the doubletransformants are readily isolated. Plants which survive the transgenicselection are grown to maturity and their seed harvested. The seeds oftransformed plants are analyzed by isolation of fatty acid methyl estersfollowed by gas chromatography to determine the fatty acid composition.

Plants expressing only the Δ⁸-desaturase will desaturate the 20:1Δ¹¹fatty acid that occurs naturally in the Arabidopsis seed to20:2Δ^(8,11). Seed harvested from plants doubly transformed with bothdesaturases will, in addition, convert the 20:2Δ^(8,11) product of theΔ⁸-desaturase plants to 20:3Δ^(5,8,11) as a result of the expression ofthe Δ⁵-desaturase. These changes will be easily detected by the fattyacid methyl ester analysis.

Example 16 A Yeast Cell Transformed with the Δ⁵- and Δ⁸-Desaturase Genesof the Invention

The cDNA portion of pJW541 (Wallis and Browse, Arch. Biochem. Biophys.365:307-316, 1999) containing the Euglena Δ ⁸-desaturase was excisedfrom that plasmid with the restriction enzymes EcoRI and SpeI. Thepurified DNA fragment representing the insert was ligated into the yeastexpression vector pYX232 (R&D Systems, Inc.) that had been prepared bydigestion with EcoRI and NheI, to give compatible sticky ends. (PlasmidpYX232 carries the marker conferring yeast prototrophy for tryptophan(TRP1 mutation), and uses the triose phosphate isomerase (TPI) promoterfor constitutive expression of the inserted DNA.) The resulting plasmid,pYX232-541, was introduced into the Saccharomyces cerevisiae strainalready harboring the Δ⁵-desaturase (pYFAT4; Watts and Browse, Arch.Biochem. Biophys. 362:175-182, 1999) plasmid that confers yeastprototrophy for uracil using a lithium acetate transformation procedure(Invitrogen). Transformants were selected simultaneously for uracil andtryptophan prototrophy. Selected colonies arising after thetransformation were inoculated into yeast minimal medium that alsolacked both uracil and tryptophan.

For analysis of activity, separate cultures were supplemented with oneof three fatty acid substrates provided as sodium salts as described(Wallis and Browse, Arch. Biochem. Biophys. 365:307-316, 1999). Afterovernight culture at 28° C., the cultures were harvested bycentrifugation and washed. Fatty acid methyl esters were prepared usingthe standard methods described in Miguel and Browse J. Biol. Chem.267:1502-1509, 1992.

Analysis by gas chromatography indicated that each substrate had beendesaturated twice. The incorporation of the three substrates varied,with more unsaturated substrates becoming a greater part of the fattyacid composition of the cells, as seen in other experiments (Wallis andBrowse, Arch. Biochem. Biophys. 365:307-316, 1999, and Watts and Browse,Arch. Biochem. Biophys. 362:175-182, 1999. For the tri-unsaturatedsubstrate 20:3Δ^(11,14,17) the 20-carbon fatty acid represented 37% ofthe total cellular fatty acid, for 20:2Δ^(11,14) the 20-carbon fattyacid level 21%, and for 20:1Δ¹¹ the 20-carbon fatty acid level reachedonly 13%. However, the activities of the desaturases were substantiallyidentical against all three substrates. Between 70 and 72% of thesubstrate was not converted, and 17 or 18% underwent a singledesaturation by only one of the enzymes. However, for each substrate,between 11 and 13% of the substrate was desaturated by both enzymesacting in concert to produce a fatty acid with two double bonds morethan in molecules of the supplied substrate.

TABLE 2 Uncon- Fatty verted One acid sub- added Fatty acid up- stratedesatu- Doubly desaturated product supplement take* # ration # # 20:3(11, 14, 17) 37 71 17 11 20:5 (5, 8, 11, 14, 17) 20:2 (11, 14) 21 70 1813 20:4 (5, 8, 11, 14) 20:1 (11) 13 72 18 11 20:3 (5, 8, 11) *as masspercent of whole cell fatty acids # as mass percent of incorporated20-carbon fatty acids

The foregoing embodiments and examples are provided only as examples andare in no way meant to limit the scope of the claimed invention.

It should be apparent to one skilled in the art that the inventiondescribed herein can be modified in arrangement and detail withoutdeparting from the scope or spirit of the invention. We claim all suchmodifications.

The references and publications referred to herein are herebyincorporated by reference.

We claim:
 1. A transformed plant, bacterial, insect, fungal, ormammalian cell expressing a non-naturally occurring recombinantpolypeptide that comprises all of: i) a cytochrome b5-like domain; ii)three His-box motifs; and iii) at least three hydrophobic regionsspanning a membrane layer twice; wherein the polypeptide: has an aminoacid sequence having at least 50% sequence identity to SEQ ID NO: 4; andis coded for by a recombinant nucleic acid molecule comprising a coreregion sequence amplifiable by polymerase chain reaction using theprimers having the sequence of SEQ ID NO: 5 and SEQ ID NO:
 6. 2. Thetransformed plant, bacterial, insect, fungal, or mammalian cell of claim1, wherein the core region sequence is a sequence amplifiable from thecDNA of a microorganism of genus Euglena by polymerase chain reactionusing the primers having the sequence of SEQ ID NO: 5 and SEQ ID NO: 6.3. The transformed plant, bacterial, insect, fungal, or mammalian cellof claim 2, wherein the polypeptide comprises an amino acid sequence atleast 70% identical to SEQ ID NO:
 4. 4. The transformed plant,bacterial, insect, fungal, or mammalian cell of claim 3, wherein thepolypeptide comprises an amino acid sequence at least 80% identical toSEQ ID NO:
 4. 5. The transformed plant, bacterial, insect, fungal, ormammalian cell of claim 4, wherein the polypeptide comprises an aminoacid sequence at least 90% identical to SEQ ID NO:
 4. 6. The transformedcell of claim 1, wherein the cell is a plant cell of a tomato, potato,yam, apple, pear, rapeseed, sunflower, peanut, soy, wheat, rice, barley,or corn plant.
 7. A non-human transgenic organism, comprising thetransformed cell of claim 1, wherein the non-human transgenic organismis selected from the group consisting of plants, bacteria, insects,fungi, and mammals.
 8. A method for creating a double bond between twocarbons in a fatty acid, comprising: contacting a fatty acid with atleast one transformed plant, bacterial, insect, fungal, or mammaliancell of claim 1; and allowing the recombinant desaturase proteinexpressed therein to create a double-bond between two carbons.
 9. Themethod of claim 8, wherein the transformed plant, bacterial, insect,fungal, or mammalian cell in a transgenic plant, bacterial, insect,fungal, or mammalian organism and the double-bond formation occurs invivo.
 10. The method of claim 8, wherein the desaturase is expressed invitro and the double-bond formation occurs in vitro.
 11. The method ofclaim 9, further comprising expressing a second desaturase in thetransformed plant, bacterial, insect, fungal, or mammalian cell.
 12. Themethod of claim 11, wherein the second desaturase has an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO: 2; (b) an amino acid sequence thatdiffers from that specified in (a) by one or more conservative aminoacid substitutions; and (c) an amino acid sequences having at least 60%sequence identity to the sequences specified in (a) or (b).